T h e COD method used in this work is a modification of the Bremner-Jenkinson method (10) and appears elsewhere (25).When referenced against the COD method, the Model DC-50 results, in the range 10-60 mg/l. C, can be shown to yield an average percent recovery of 93.0% (and a standard deviation of 11.6%). The slightly low average recovery together with the somewhat high standard deviation is indicative of the approximate nature of the COD method (Le., the assumption is made that the summation of the various oxidation/reduction processes, in the presence of the dichromate and acid, is equivalent to a +4 valence change for each organic carbon atom present). Comparison of the organic and total carbon results (Table 111) shows that the sediment sample identified as No. 7 is calcareous. T h e inorganic carbon computed from the Model DC-50 results is 25.0 mg/g C. The corresponding result computed from the LECO and COD results is 26.2 mg/g C. Table IV compares the ratio of carbohydrate to organic carbon in the samples. The ratios vary from 0.16 to 0.95 with a mean of 0.54. Such ratios, when considered with the total organic carbon content, could presumably be a valuable aid in the classification of sediments, since sedimentary deposits from various sources (i.e., decaying vegetation, sewage, pulp and paper wastes, packing house wastes, and food processing wastes, etc.) are likely to have characteristic carbohydrate/organic carbon ratios. I t is evident from the results of this work that a carbon analyzer (Model DC-50) using the reductive pyrolysisflame ionization technique is suitable for the determination of both total and organic carbon in sediments. An added advantage is that the determinations require less than 5 min. In addition, the use of the same, or similarly prepared, suspensions in the determinations allows for a direct basis of comparison of the total carbon, organic carbon, and carbohydrate levels of a particular sediment. Since the detection limit of all three tests is 1 ppm carbon, the sensitivities
obtainable, when the concentration of the suspension is 3000 ppm, are 0.3 mg/g carbon.
LITERATURE CITED (1) J. G. Konrad, D. R . Keeney, G.Chesters, and K. L. Chen, J. Water Pol/ut. Contr. Fed., 42, 209 (1970). (2) D. G . Ballinger and G. D. McKee, J. Water Pollut. Contr. Fed., 43, 216 (1971). (3) G. C. Bortleson and G. F. Lee, Environ. Sci. Technol., 6 , 799 (1972). I S. E. Lindberg and R. C. Harris, Environ. Sci. Technol., 8, 459 (1974). I S. Fliesher, Arch. Hydrobiol.,7 0 , 302 (1972). I D. Liu. P. T. S. Wong, and B. J. Dukta. WaterRes., 1, 741 (1973). 1 L. E. Allison, Roc. SoilSci. SOC.Am.. 24, 36 (1960). B. A. Stewart, L. K. Porter, and W. E. Beard, Proc. Soil Sci. SOC.Am., 28. 366 (1964). J. L. Young and M. R. Lindbeck, Proc. Soil Sci. SOC. Am., 28, 377 (1964). J. M. Bremner and D. S.Jenkinson, J. Soil Sci., 11, 394 (1960). C. E. Van Hall and V. A. Stenger, Anal. Chem., 39,503 (1967). Y. Takahashi, R . T. Moore, and R . J. Joyce, Am. Lab., 4, 31 (1972). R. B. Schaffer. C. E. Van Hall, G. N. McDermott, D. Barth, V. A. Stenger, S. J. Sibester, and S. H. Griggs. J. Water Pollut. Conk Fed., 37, 1545 (1965). N. R. McQuaker, Report No. 7416, Chemistry Laboratory, Water Resources Service, Province of British Columbia, 1974. M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith, Anal. Chem., 28, 350 (1956). V. Y. Artem'yev. Oceanology, 9 203 (1969). Dohrmann-Envirotech Corp., Mountain View, CA, DC-50 Total Organic Carbon Analyzer Instruction Manual (1972). P. R . Hesse, "A Textbook of Soil Chemical Analysis". John Murray (Publishers) Ltd., London, 1971, p 14. P. R. Day, "Particle Fractionation and Particle Size Analysis". in "Methods of Soil Analysis, Part One", C. A. Black, Ed., Agronomy No. 9, 1965, p 545. R . D. Guthrie and J. Honeyman. "An introduction to the Chemistry of Carbohydrates", Oxford University Press, 1964, p 107. J. E. Modzeleski, W. A. Laurie, and B. Nagy, -. Geochim. Cosmochim Acta, 35, 825 (1971). M. V. Cheshire and C. M. Mundie. J. Soil Sci., 17, 372 (1966). N. Handa. J. Oceanogr. SOC.Jpn., 22, 1 (1966). L. M. Lavkulich, "Methods of Soil Analysis", Pedology Laboratory, Department of Soil Science, University of British Columbia, 1974, p 43. N. R. McQuaker, "The Chemical Analyses of Waters, Wastewaters, Sediments and Biological Materials", Chemistry Laboratory, Water Resources Service Province of British Columbia, 1974, p 49.
RECEIVEDfor review December 17, 1974. Accepted February 18,1975.
Determination of Cadmium in Fish Tissue by Flameless Atomic Absorption with a Tantalum Ribbon E. R. Blood and G. C. Grant Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284
Cadmium is increasingly recognized as a n important environmental pollutant with toxic effects on human and animal life a t relatively low levels (1-3). Environmental concentrations of cadmium are of serious concern because cadmium accumulates in the human body throughout lifefrom approximately 1 pg a t birth to about 30 mg with about one third to be found in the kidneys ( 4 ) . Based on animal studies, cadmium is preferentially retained by the kidney and liver (2). In view of the known accumulation of cadmium in biological tissues, a detailed study was begun to determine the rate of uptake in the common bluegill (Lepornis rnacrochirus Raf.) exposed to known amounts of cadmium in a carefull controlled aquatic environment. An important objective was to evaluate the relative rates of uptake in vital organs including: heart, skin, muscle, gut, gill, kidney, liver, 1438
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8. JULY 1975
and bone. A recent study of the toxicity of ZnS04 to rainbow trout dealt with only acute toxicity as measured by fish mortality ( 5 ) .Another study examined the chronic toxicity of copper, cadmium, and zinc mixtures a t sub-lethal concentrations to the fathead minnow using mortality, physical characteristics, and reproduction as bioassay methods (6). While these studies increase our understanding of biological effects for relatively concentrated heavy metal pollutants in aquatic systems, they provide no evidence for the actual rate of accumulation of toxic metals nor the distribution of these in vital organs. Moreover, only rarely in natural waters do the concentrations of toxic metals attain the levels used in most acute and chronic bioassay studies. Thus, experimental evidence for heavy metal accumulation and distribution in organisms exposed to environmentally realistic levels of heavy metal pollutants in
Table I. Instrumental Parameters
1
Model 153 A . A . Spectrophotometer
Mode Hollow cathode A B Lamp current A B P. M. voltage Slit width Wavelength
A-B (Background Correction) Cadmium H, continuum 3.5 mA 20mA 700 V 320 l m 228.8 nm
50 m a CI
cp
602
517
Model 355 Flameless Sampler
Mode Purge gas Gas flow r a t e D R Y setting D R Y time A N A L Y Z E setting
Automatic Argon 10 SCFH
A2
170 "C (2.5 turns) 140 seconds 1600 "C (7.0 turns)
natural waters should make possible more reliable and general predictions for the long term effects of such pollutants. One recent method for t h e determination of lead in fish using atomic absorption with a graphite furnace has been reported (7), but was limited t o the muscle. T h e present work is a study of analytical methods for t h e analysis of cadmium in fish tissue by atomic absorption spectrophotometry using t h e higher sensitivity afforded by flameless atomization of t h e sample.
EXPERIMENTAL All analyses were performed on an Instrumentation Laboratory (IL) Model 153 Double Beam Atomic Absorption Spectrophotometer, modified electronically to allow subtraction of non-specific absorption from a hydrogen continuum placed in channel B as previously described by Hwang (8).Samples were dried and vaporized on the tantalum ribbon of an IL Model 355 Flameless Sampler. Instrumental parameters were optimized according to procedures previously described (8, 9). AA signals were monitored with a Heath EU2OB recorder. All solid reagents and concentrated acids were ACS reagent grade and were used without further purification. Distilled water, which had been passed through a mixed bed ion exchange column, was used throughout this work. Glassware, polypropylene test tubes, and polyethylene storage bottles were standard laboratory equipment. Glassware was cleaned with concentrated containing KMn04 followed by repeated water rinses. Polypropylene test tubes were soaked at least 24 hours in 5 w/v% Triton X-100 solution followed by water rinses. Gilmont micrometer syringes were used. Hatchery-obtained fish were approximately the same size and weight (5-10 cm, 3-5 g). Twenty-eight fish were placed in all-glass aquaria containing reconstituted hard water (30 mg MgSOd., 30 mg CaS04/1., 48 mg NaHC03/1., and 2 mg KCl/l.). After two weeks of acclimation, sufficient CdClz was added t o obtain 3 fig Cd/ml in each tank. Fish were removed at set intervals throughout the twoweek experiment and frozen in plastic bags until analysis. T w o wet acid digestion mixtures were compared for the analysis of fish tissue. In the first, 1 ml of the acid mixture (3 parts concentrated "03 by vo1ume:l part concd HzS04:l part concd HC104) was placed together with a weighed sample (1to 100 mg depending on the organ used) in a covered 3.5-ml polypropylene test tube for 2 hours at 74 "C in a water bath and finally, dilution to 25 ml. The second procedure, reported recently ( I I ) , was initiated by addition of 1 ml of concentrated "03 to the sample and heating for 15 minutes at 80-90 O C followed by addition of 1 ml of 10%H202 and heating for an additional 15 minutes. For analysis, the digested sample was diluted to 25 ml in a volumetric flask and 20-fil aliquots were removed immediately for analysis with an Eppendorf pipet to prevent adsorption of cadmium on the glass surfaces. Since peak heights observed for cadmium standards decreased approximately 20% during the lifetime of a tantalum ribbon (50-100 aliquots) and varied from ribbon to ribbon, no attempt was made to use a standard curve. Instead, the
20 6 t
1.1 15 I %I
52.3
Figure 1. Effect of background correction on cadmium peak height
Repetitive vaporization of 2 0 4 aliquots dried at 170 O C for 140 sac and atomized at 1600 O C . Each trace is 160 seconds in duration. A T , AP. and A3 are 5.00 ng Cd/ml ( 4 % acid mixture) in normal mode (without background correction);01, 0 2 , and B3 are the same solution with background correction; C1 and C2 are bluegill skin in the acid digest without background correction; D is the same bluegill skin sample with background correction unknown concentration was calculated from the ratio of peak heights of a cadmium standard chosen to have a similar peak height. The normal analysis procedure was to analyze two aliquots of standard, four of the sample, followed by two of the standard. Any observed irregularities in the standards resulted in the analysis of additional aliquots.
RESULTS AND DISCUSSION Instrumental parameters optimized for the analysis of fish tissue are listed in Table I and were chosen for highest reproducibility at a small sacrifice of sensitivity. An increase of t h e atomize temperature t o 1700 "C gave increased sensitivity but with relative standard deviation (rsd) in the peak height two t o three times larger. Accumulation of matrix components or non-volatilized cadmium was counteracted by briefly heating t h e ribbon t o 2400 O C between aliquots. A shorter dry time would reduce considerably the total analysis time but would not allow complete volatilization of t h e strongly acid matrix prior to atomization. Twenty mA current for the hydrogen continuum provided adequate background correction a t 228.8 nm for cadmium while extending the life expectancy of the hydrogen lamp. Figure 1 shows the typical effects of using the background correction mode on 5.0 ng Cd/ml aqueous cadmium standard in t h e diluted acid mixture (1:25). Each peak is a 2 0 4 aliquot from a n Eppendorf pipet. T h e background correction mode for standards typically gave: 1)absence or sharply reduced matrix forepeak, 2) increased noise level, 3 ) reduced precision (higher rsd) and 4) approximately a 5% decrease in mean peak height. In a n effort t o improve the relative precision of the analyses, the use of a micrometer syringe was investigated. Analysis of successive aliquots of a 5.0 ng Cd/ml solution gave 1.5 and 3.0% rsd for the micrometer and Eppendorf pipets respectively (10 aliquots). However, significantly, such precision with the micrometer syringe was attainable only with very careful technique because significant loss of peak height was observed whenever the solution remained ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1439
Table 11. Recovery of Cadmium after Acid Digestion of S t a n d a r d s
Table 111. Comparison of Cadmium Found in Bluegill Tissue after Acid Digestion
Recover),
m0j Sample
Bovine Liver (NBS SRM 1577) 0.27 i 0.04 ppmQ 0.500 pg Cd/ml
H202
ppm Cdb
HhO3 HzSOd,HC104
dtqestion
digestion
85 (0.23 ppm) 88.6 79.2 83.5 87.9
93 (0.25 ppm) 99.9 95.7 93.1 96.1
-
Samplea
(tfi03/H2@2)
Muscle Muscle Skin
0.91 1.41 8.6
ppm Cd ( H 4 0 j / rsd,
H2504’HCl@h)
rsd,
’,
9.9 7.6
1.25 15.9 2.87 14.5 3.2 10.9 7 .O a A portion of the wet homogenate from a ground glass homogenizer. ppm Cd is pg Cd per gram of wet tissue.
-
Mean 84.8 Mean 96.2 a ppm Cd is pg Cd per gram of dried sample
sample was carefully homogenized before weighing portions for the digestions so that the differences in the ppm Cd found reflect real differences in the digestion step or matrix interferences in the subsequent analysis. Although the relative precision was better for the HN03/Hz02 method, the percent recovery was always smaller indicating sysinside the syringe for even a few minutes, indicating rapid tematic errors. Additionally, although the HN03/H202 diabsorption of cadmium on either the glass or needle which gestion appears to require less operator time for digestion, is consistent with other recent work (IO). The increased it actually requires more time when multiple samples are convenience of use, the fri.edom from adsorption, and the run because it must be watched constantly whereas the freedom from cross-cuii~a,nination(using disposable tips) were judged to be well worth the small loss in relative preHN03/HzS04/HC104 digestion can be left unattended. Furthermore, several samples foamed over the test tube cision. Also, in agreement with these adsorption studies (IO), peak heights for freshly diluted 1 or 5 ng Cd/ml stanupon addition of HzOz, necessitating repetition of the experiments. For these reasons, the HN03/H&04/HC104 didard solutions were (within day to day reproducibility) the same as solutions aged for periods of several months in gestion method is recommended for fish tissue. The concentrated acid mixtures were found to have 7.5 and 10.5 ng polyethylene bottles. Nevertheless, standards were usually Cd/ml for HN03/H202 and HN03/HzS04/HC104, respecfreshly diluted every few days from more concentrated tively, which was low enough for most of the samples anastock solutions and contact time with glass vessels was lyzed that expensive ultrapure acid reagents could be minimized. avoided. All sample and standard peak heights were corTypical results for background correction in the various fish organs in the acid digest are also shown in Figure 1. rected for the contribution of the acid blank, which was 0.41 ng Cd/ml (HN03/HzS04/HC104) after dilution to 25 Cadmium peaks for fish samples were typically 10-20% ml. lower using background correction indicating a significant Typical results for eight organs of three bluegills digestnonspecific absorption and/or light scattering during the ed with the HN03/HzS04/HC104 acid mixture are given in analyze cycle. Background correction was therefore rouTable IV. In the case of the heart, kidney, and liver, the tinely used. During the initial phases of this work, 10-ml glass vials sample weight reflects the entire organ which was excised with snap caps were used as containers for the wet acid di- without connective tissue. Samples of the other organs gestions. In agreement with other recent work ( 3 ) , reprowere chosen from representative portions. The analyte conducibility was very poor with percent recoveries of stan- centrations after correction for the acid mixture blank dards as low as 74%; therefore, all further acid digestions (0.41 ng/ml) ranged from 0.1 to 9 ng/ml and were comparawere done with polypropylene test tubes. ble to blank levels only for the heart and muscle. Since 20Results for samples digested by both procedures are microliter aliquots of a 1 ng/ml solution exceed the detecgiven in Table 11. Analysis of National Bureau of Standards tion limit for cadmium by this technique ( 2 X gram) by a factor of ten and since the amount of cadmium per alibovine liver (Standard Reference Material No. 1577) by quot could be further increased by increasing the size of the both digestion procedures gave results within the error tolerance given but the HN03/H202 method gave only 85% re- aliquot to 50 pl, dilution of the digested sample to a smaller volume or increasing the sample size, instrumental sensicovery as compared with 95% for the HN03/HzS04/HC104 tivity is more than adequate for analysis of all organs studmethod in good agreement with the percent recovery of a 0.500 pg Cd/ml standard run through the identical diges- ied here. For analysis of still lower levels than detected tion procedures. Other workers (11) have reported 96% re- here, the use of specially purified reagent acids will result covery (0.26 ppm Cd) in SRM 1577 by the same techniques in an acid blank concentration of cadmium reduced by a factor of ca. 10. using the HN03/Hz02 digestion procedure, which implies Comparison of the relative standard deviations in Table that higher recovery of cadmium can be obtained with this IV for each organ with those for aqueous standards a t comacid mixture, although all of these results lie within the error bar given by NBS. Although incomplete decomposi- parable concentrations indicates a loss in precision due to the matrix remaining after acid digestion only for the skin, tion of organically bound cadmium in our digestions could kidney, and bone. The loss in precision for the heart and account for less than 100% recovery, the same percentage muscle is attributable to nearness of the sample concentrarecovery of cadmium was found after digestion of aqueous standards in each acid mixture, indicating that partial de- tion to the acid blank for the reagent acid used. Examinacomposition of organic matter alone in the H N 0 3 / H ~ 0 2 tion of the last column in Table IV, comparing organs from mixture is not an adequate explanation for the loss of cad- three fish exposed to the same amount of cadmium, reveals that reproducibility is far worse between fish than could be mium. This point was not pursued further in this work. accounted for by the precision of the method or the accuraAdditional comparison of the two digestions is possible cy of analysis (from the bovine liver data). Thus, the variausing the data in Table I11 for muscle and skin. The muscle 1440
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
Table IV. Cadmium in Fish Exposed to 3 ppm CdClz for O n e Week Sample 1-1I.1,311
\%
Heart
mq
ppm C d a ( c a c h fish)
Absolute btd dcv
Re1 std di\, '
1.52 6.34 5.62 1.61 1.07 1.26 0.20
0.18 0.57 1.01 0.24 0.13 0.38 0.02
12 9 18 15 12 30 10
0.075 5.25 0.61 5.02
0.02 0.11 0.02 0.10 0.33 0.45 0.56 0.37 0.79 0.33 0.20 0.21 0.81 0.34 0.60 0.04
27 2 3 2 5 7 60 9 35 11 4 4 12 15 23 12
1.8 2.1 2.4 22.3 48.1 17.8 45.1 85.0 29 .O 34.3 26.6 20.6 10.6 15.1 9.6 17.8 9.7 10.8 19.9 38.0 17.7 8.9 4.9 4.8
Skin
Muscle
Gut
Gill
Kidney
Liver
Bone
a
LL(jllt,
...
6.67 6.49 0.94 4.13 2.26 3.04 4.91 5.18 6.71 2.28 2.59 0.32
...
...
4vLragr ppm Cd
Re1 std d e \ ,
4.5
58
1.3
21
0.14
51
3.6
72
4.7
69
3.1
30
5.6
17
1.7
71
L
ppm = p g Cd per g of wet tissue
tion in cadmium levels is attributed primarily to physiological differences between fish. Similar variations in the cadmium content of human liver from different portions of the same liver have been attributed to the heterogeneous distribution within the organ (11). In conclusion, flameless atomic absorption with background correction has been shown to have adequate sensitivity and reproducibility for the analysis of eight fish organs to permit detailed studies of uptake and distribution of cadmium in fish exposed to elevated levels in aquatic systems. LITERATURE CITED (1) D. Lee, Ed.. "Metallic Contaminants and Human Health", Academic Press, New York, 1972. (2) L. Friberg, M. Piscator, and G. Norberg, "Cadmium in the Environment", CRC Press, Cleveland, 1971. (3) J. McCauil, Environment, 13, 3 (1971).
(4) D. B. Louria. M. M. Joselow, and A. A. Browder, Ann. intern. Med.. 78, 307 (1972). (5) J. F. De L. G. Solbe. Water Res., 8, 389 (1974). (6)J. G. Eaton, Water Res., 7, 1723 (1973). (7) G. K. Pagenkopf, D. R. Neuman, and R. Woodriff. Anal. Chem., 44, 2248 (1972). (8)J. Y. Hwang. P. A. Uiiucci, and C. J. Mokeler, Anal. Chem., 45, 795 (1973). (9) J. Y. Hwang, C. J. Mokeler, and P. A. Ullucci. Anal. Chem., 44, 2018 (1972). (IO) A. W. Struempler, Anal. Chem., 45, 2251 (1973). (11) P. A. Ullucci and J. Y. Hwang, Talanta, 21, 745 (1974).
RECEIVEDfor review December 26, 1974. Accepted March 18, 1975. P a r t of this work was supported by NSF Grant GY-9610 under the Student Originated Studies program. P a r t of this paper was presented a t the 7th Materials Research Symposium on Accuracy in Trace Analysis, National Bureau of Standards, Gaithersburg, Md., in October 1974.
Determination of Trace Bismuth in Copper by Hydride Evolution Atomic Absorption Spectrophotometry Michel Bedard and J.
D. Kerbyson
Noranda Research Centre, Pointe Claire, Quebec, Canada
The determination of bismuth has importance in commercial electro-refined copper, because of the harmful effects of this impurity on some physical and electrical properties. Typical levels are 0.1-0.4 pprn Bi, and existing analytical methods comprise spectrography, extraction-spec-
trophotometry, and extraction-atomic absorption techniques, Spectrography provides one of the most widely used methods for the rapid and sensitive determination of bismuth in copper ( I ) . The detection limit is 0.1 ppm, and ANALYTICALCHEMISTRY. VOL. 47, NO. 8, JULY 1975
1441
